Alcohol consumption and chronic infection with the human hepatitis B virus (HBV) are both major causes of liver diseases worldwide. Primary liver cancer (hepatocellular carcinoma, HCC) is the fifth most common cancer in the world; approximately 50% of HCCs are associated with chronic HBV infections. Complications of chronic HBV infections account for about 1 million deaths each year. The combination of a chronic HBV infection and alcohol consumption causes a more rapid progression of liver injury and HCC as compared to chronic HBV infections or alcohol consumption alone. Understanding how chronic HBV infections and alcohol consumption lead to the development of HCC is important for defining mechanisms of hepatocyte transformation and developing therapies for preventing HBV- and alcohol-associated HCC. My proposal focuses on the effect of the X protein (HBx) of HBV and alcohol on mitochondrial physiology and apoptosis. We have developed methods for isolating and culturing primary rat hepatocytes, thereby generating a biologically relevant system in which to conduct our studies. We will use this system to address the following specific aims: (1) we will study the interaction of HBx with mitochondria, (2) we will assay the effect of HBx and/or alcohol on the regulation of mitochondrial calcium stores, regulation of the mitochondrial permeability transition pore and regulation of mitochondrial membrane potential and (3) we will analyze how these alterations of mitochondrial physiology affect apoptosis by monitoring various markers of apoptosis such as cytochrome c release from the mitochondria and activation of caspases. The long-term goal of these studies is to generate a mechanistic understanding of the effect of HBx and alcohol on mitochondrial and hepatocyte physiology in order to better understand the signaling pathways that are activated in the development of HCC. We hypothesize that HBx and alcohol regulation of mitochondrial activities will activate apoptotic pathways in hepatocytes and that, when combined, HBx and alcohol will cause a stronger induction of apoptosis. Alcohol consumption and chronic HBV infection cause a high turnover rate of hepatocytes; therefore, in a regenerating, diseased liver, the eventual selection of hepatocytes resistant to apoptotic signals may occur, contributing to the development of HBV- and alcohol-associated HCC. The use of normal hepatocytes provides a more biologically relevant system than previous studies in immortalized or transformed cells, allowing a more sophisticated understanding of the molecular mechanisms contributing to the development of liver cancer. [unreadable] [unreadable] [unreadable]